Lone IN et al. (2013), Binding of NF-κB to nucleosomes: effect of tran...

XB-ART-53500

PLoS Genet 2013 Jan 01;99:e1003830. doi: 10.1371/journal.pgen.1003830.

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Binding of NF-κB to nucleosomes: effect of translational positioning, nucleosome remodeling and linker histone H1.

Lone IN , Shukla MS , Charles Richard JL , Peshev ZY , Dimitrov S , Angelov D .

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NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.

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Species referenced: Xenopus
Genes referenced: arhgef7 h2ac21 h2bc21 kidins220 nf1

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	Figure 1. Characterization of the reconstituted nucleosomes.(A) Schematics of the reconstituted nucleosomes. The canonical NF-κB site was inserted in the 255 bp 601 DNA fragment either at the dyad of the nucleosome (601_D0 DNA) or at the nucleosomal end (601_D7 DNA) or in the linker DNA (601_D8 DNA); bold lines, free DNA arms; dashed line, core particle region. The vertical black line represents the dyad. The NF-κB binding sites (BS) are depicted by the red line. The length of each region is shown on top of the constructs. The very bottom schematics shows the location of the NF-κB binding site inserted in the 5S rDNA fragment of Xenopus borealis used for nucleosome reconstitution. (B) Electrophoretic analysis of the indicated purified recombinant histones and histone octamer. (C) Electrophoretic analysis of purified recombinant NF-κB (p50); M, molecular marker; p50, the p50 subunit of NF-κB. (D) Nucleosome reconstitution check by 5% native PAGE. (E) •OH radical and (F) DNase I footprinting of free 601 DNA and the indicated reconstituted nucleosomes.
	Figure 2. NF-κB binds specifically at the nucleosomal ends, but not at the nucleosomal dyad.(A) Upper panel: EMSA of NF-κB binding to naked 601_D7 DNA (left) or to 601_D7 nucleosome (right). Naked 32P-end labeled 601_D7 DNA or nucleosomes were incubated with increasing amount of NF-κB (1.5 fold serial dilution of NF-κB was performed starting with saturating amounts of 100 nM for naked DNA and 400 nM for nucleosome). The aliquots of the reaction mixtures were run on a 5% native PAGE. The positions of free DNA and nucleosomes are indicated, “cplx.” represents NF-κB – DNA/nuc complexes; lower panel: UV laser footprinting patterns of the NF-κB-DNA and NF-κB-nucleosomes complexes. The respective remaining mixtures were irradiated with a single 5 nanoseconds UV laser 266 nm pulse (E pulse∼0.1 J/cm2), DNA was purified from the samples and then treated with Fpg glycosylase. The cleaved DNA fragments were separated on 8% sequencing gel and visualized by autoradiography; (*), NF-κB footprint at the high-affinity NF-κB binding site; (♦), NF-κB footprints at low-affinity sites. A schematic presentation of the nucleosomes is shown on the right side; the double headed arrow indicates the nucleosomal dyad. (B) UV laser footprinting of NF-κB bound to either naked 601_ D7-DNA (lanes 1 and 2) or to 601_D7-nucleosome (lanes 3 and 4) repeated with saturated amounts of NF-κB as indicated. (C) Same as (B), but for naked 601_D0 DNA and 601_D0-nucleosomes. Note the absence of specific NF-κB footprint at the dyad in the case of the nucleosome.
	Figure 3. Binding of NF-κB to remodeled and slid nucleosomes.(A) Nucleosomes, RSC-remodeled nucleosomes (remosomes), slid nucleosomes and naked 601_D0 DNA were incubated with the indicated amount of NF-κB and separated on a 5% native PAGE. The positions of the different particles are shown on the left part of the gel. (B) UV laser footprinting of the indicated distinct NF-κB bound particles. The experiment was carried out as described in Figure 2. The NF-κB binding site is shown as vertical black line and the black arrow indicates the nucleosomal dyad; M, 10 bp DNA molecular marker (C) top, “zoom” of the NF-κB binding region from the footprints shown in (B); bottom, scan of the footprints; red, in presence NF-κB; green in absence of NF-κB.
	Figure 4. Dilution driven H2A–H2B dimer eviction allows binding of NF-κB to Nucleosome Core Particle.(A) 152 bp DNA fragment derived from X. borealis somatic 5 S RNA gene containing single NF-κB site near the dyad NF1 (53–56) was amplified by PCR and uniquely 3′ end labeled with α-32P by Klenow. Nucleosomes were reconstituted on this labeled fragment as described previously. The DNA and nucleosomes at a concentration 40 nM were incubated with increasing amounts of NF-κB as indicated to allow the formation of stable complexes which were subsequently irradiated by a single high intensity UV laser pulse (E pulse∼0.1 J/cm2). The formation of complexes was checked by EMSA (upper panel, DNA lane 1–3, nucleosomes lane 4–9), the positions of free DNA and nucleosomes are indicated, “cplx.” represents NF-κB – DNA/nuc complexes. The samples were split into two parts, DNA was purified and treated with Fpg glycosylase to cleave 8-oxoG (represented by ▸,lower panel, DNA lane 1–3 and nucleosome lane 4–9) and with T4 endonuclease V to cleave CPDs (represented by ◊, DNA lane 1′–3′ and nucleosome lane 4′–9′). The cleaved DNA fragments were visualized by 8% sequencing gel. (B) The same 152 bp 5S fragment was 5′ end labeled with γ-32P by T4 polynucleotide kinase and used for nucleosome reconstitution. DNA and nucleosomes, at 10 nM final concentration, were incubated with increasing amounts of NF-κB as indicated to allow the formation of complexes. The assembly of the complexes was checked by EMSA (upper panel, DNA lane 1–5, nucleosomes lane 1′–5′). The samples were irradiated with a single high intensity UV laser pulse (E pulse∼0.1 J/cm2), treated with a mix of Fpg glycosylase and T4 endonuclease V to cleave both the 8-oxoG (▸) and CPDs (◊). Finally, the cleaved products were visualized by 8% sequencing gel (DNA, lane 1–5; nucleosomes lane 1′–5′). The NF-κB binding sites (vertical bold lines) and the NF-κB recognition sequences are shown. The arrows designated the nucleosomal dyad.
	Figure 5. NF-κB displaces H1 from the chromatosome and binds to its recognition sequence.(A, upper panel), schematics of the substrates used in each experiment. (A, lower panel), EMSA of the binding of NF-κB to depicted substrates. The bottom strand of the 255 bp 601_D8 DNA (Supplementary figure S1) was uniquely 5′-end labeled by 32-P and used to reconstitute centrally positioned nucleosomes. Chromatosomes were assembled by using the NAP-1/H1 complex to properly deposit H1 on the nucleosome in H1/nuc ratio of ∼1.5. The first two panels show the NF-κB-DNA (lanes 1–4) and NF-κB-nucleosome (lanes 5–8) complexes formed upon incubation with increasing amount of NF-κB. The last panel illustrates both the interaction of chromatosomes with the indicated increasing amount of NF-κB (lanes 1′–5′) and the deposition of H1 on the already assembled (at increasing NF-κB concentration) NF-κB nucleosome complexes (lanes 6′–9′). (B) UV laser (upper panel) and •OH (lower panel) footprinting of the NF-κB binding region of NF-κB-DNA and distinct NF-κB-nucleosome complexes.

References [+] :

Adams, Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. 1995, Pubmed